Methods for determining the metabolism of sugars and fats in an individual

ABSTRACT

Provided herein are methods for determining the metabolism of one or more sugars and/or fatty acids, and applications thereof. Such applications include determining the rate of glycogen synthesis and glycolysis, which are believed to be early markers for predicting elevated risk of diabetes and cardiovascular disease. Other applications include methods for screening drugs that effect sugar and/or fatty acid metabolism. The methods are useful for at least partially characterizing drugs for desirable or undesirable (toxic) characteristics. Drugs that are at least partially characterized using the methods of the invention can then be further developed in pre-clinical testing and clinical trials. Such drugs may be found to be useful in treating obesity, diabetes, cardiovascular disease, and other disorders of metabolism.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 60/423,964 filed Nov. 4, 2002, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of sugar and fatty acidmetabolism. In particular, methods for determining the metabolism of oneor more sugars or fatty acids in living organisms, including humansubjects, are described.

BACKGROUND OF THE INVENTION

Utilization of nutrients is key to many diseases, including obesity,insulin resistance/diabetes mellitus, hyperlipidemia, and others. Thecapacity to oxidize dietary fat relative to the tendency to storeingested fat, for example, is considered to be a central determinant ofsusceptibility to dietary fat-induced obesity. Similarly, the capacityto store or oxidize dietary glucose is a key element in insulinresistance and glucose intolerance/diabetes. Tools for assessing thefate of nutrients in the body in living organisms have lagged behind,however. Currently available tools suffer from many limitations.

The oral glucose tolerance test (OGTT) is widely used in medicalresearch and clinical medicine for assessing insulin sensitivity oftissues. The principle of the OGTT is that uptake of glucose from bloodby tissues, along with suppression of release of endogenously producedglucose into blood from tissues, is reflected in the clearance rate ofan exogenous glucose load from the bloodstream. This approach is crude,however, and no information is generated about the specific metabolicfate or consequences of the glucose administered. As a result, noinformation is generated about the mechanisms underlying impairedglucose tolerance. Though widely used in clinical practice, the OGTT isof limited utility.

Fat tolerance testing has a similar basis and similar limitations asOGTT. The fat tolerance test measures the uptake of fatty acids fromblood by tissues. This approach is also crude, and

Fat tolerance testing has a similar basis and similar limitations asOGTT. The fat tolerance test measures the uptake of fatty acids fromblood by tissues. This approach is also crude, and gives no informationabout the specific metabolic fate or consequences of the fatadministered. As a result, no information is generated about themechanisms underlying impaired fat tolerance. Fat tolerance testing hasmostly been used to assess the clearance of dietary fat from blood incontext of evaluating hyperlipidemia. Fat tolerance testing is nothelpful for assessing sensitivity to high fat-induced obesity.

Indirect calorimetry (IC), or the measurement of fuel oxidation based onrespiration, is useful for whole body studies. IC, however, is expensiveand requires complex equipment for small animal studies. Also, IC onlyreveals the net oxidation of fuels in the whole body, without revealingmore details concerning the fate of individual fuels in the tissues.

Insulin/glucose clamps and other intensive approaches are of limitedpractical utility in clinical practice or broad-based drugscreening/discovery, due to their labor-intensive nature. Physiologicrelevance is often also uncertain, since the procedures used (e.g.intravenous glucose infusion at high rates) do not mimic normalphysiologic intake of these nutrients.

The most direct approach is by use of isotopic techniques. These havebeen highly problematic, however. The oxidation of ¹³C- or ¹⁴C-labeledglucose or fatty acids to ¹³CO₂ or ¹⁴CO₂ has been used as a marker oftissue oxidation (1-3). The references cited herein are listed at theend of the specification before the claims. The serious flaws with thisapproach have been discussed previously (4). In brief, recovery oflabeled CO₂ is a highly variable and unreliable index of tissueproduction of CO₂, due to re-utilization/exchange pathways of ¹³CO₂ or¹⁴CO₂. Yield of labeled CO₂ generated oxidatively in tissues can be aslow as 20%, or as high as 80% (1-4).

The most common risk factor setting for cardiovascular disease is theso-called syndrome X or multiple risk factor syndrome (15) wherein anindividual exhibits the combination of obesity, hypertension,hyperlipidemia, and glucose intolerance or diabetes. This syndrome isnow widely believed to be tied together pathogenically by insulinresistance, defined as lower-than-normal sensitivity of tissue to theeffects of insulin on glucose metabolism (15).

A primary component of tissue insulin resistance is impairment of theefficiency and rate of skeletal muscle and adipose tissue uptake andmetabolism of glucose in response to insulin exposure. One component oftissue glucose metabolism is storage as glycogen; the main alternativepathway for glucose metabolism in a tissue is glycolytic metabolism,leading to oxidation or other fates (FIGS. 2 and 3). Both the storage(non-oxidative) and glycolytic (oxidative) pathways are impaired ininsulin resistant tissues, such as skeletal muscle (15).

Because the insulin resistance syndrome is so common—indeed is the mostcommon medical abnormality in contemporary Western populations—areliable laboratory test for diagnosing and monitoring insulinresistance has long been a very high priority. Various commentators havestated that a clinical marker of insulin resistance would be a “holygrail in the fields of modern diabetes and cardiovascular disease” (C.Kahn, M. D., Director of Scientific Sessions, American DiabetesAssociation, October 2003). The availability of a clinical test forinsulin resistance would affect not only patient care but also wouldallow drugs to be developed specifically to treat insulin resistance.

Unfortunately, no current laboratory test is a reliable measure ofinsulin resistance. Serum insulin concentrations are highly variablefrom assay to assay and are influenced by insulin clearance as well astissue sensitivity to insulin. Other measures, such as bloodtriglyceride concentration, fasting glucose concentration, oral glucosetolerance, body mass index, waist-to-hip ratio, etc. correlate poorlywith clinical insulin sensitivity (as measured by a labor-intensiveresearch test, such as the insulin-glucose clamp technique; see Ref.15).

A technique for quantifying glucose metabolism by tissues—in particular,glycolysis and/or glycogen storage of a glucose load—would thereforehave enormous impact on medical practice and drug trials.

SUMMARY OF THE INVENTION

In order to meet these needs, the present invention is directed tomethods of determining the metabolism of one or more sugars or fattyacids, and uses of the methods in diagnosis and testing, and kits fordetermining the metabolism of one or more sugars and fatty acids.

In one format the invention disclosed herein represents a reliablemeasure of insulin resistance, and reveals tissue insulin sensitivity orresistance in an individual. Use of the methods disclosed herein allowsdiagnostic classification of patients (for decisions regardingrisk-factor interventions), clinical monitoring of treatments intendedto improve insulin sensitivity and reduce insulin resistance (such asthe thiazolidinedones or metformin), and clinical development of newagents to treat insulin resistance (as an end-point or biomarker of drugeffect).

In one variation, the invention is directed to a method of determiningmetabolism of one or more sugars or fatty acids in an individual, wherethe method includes (a) administering one or more compositions of one ormore ²H-labeled sugars or ²H-labeled fatty acids to an individual; (b)obtaining one or more bodily tissues or fluids at one or more times fromthe individual; and (c) detecting the incorporation of the ²H from the²H-labeled sugars or ²H-labeled fatty acids into water to determine thesugar or fatty acid metabolism in the individual.

In another variation, the one or more compositions include ²H-labeledglucose. In another variation, the one or more compositions include[6,6-²H₂]glucose, [1-²H₁]glucose, [3-²H₁]glucose, [2-²H₁]glucose,[5-²H₁]glucose, or [1,2,3,4,5,6-²H₇]glucose.

In another variation, the one or more compositions are administeredorally, by gavage, intraperitoneally, intravenously, or subcutaneously.In a further variation, the one or more compounds are administeredorally.

In another variation, the individual is a mammal. In a furthervariation, the mammal is chosen from humans, rodents, primates,hamsters, guinea pigs, dogs, and pigs. In a still further variation, themammal is a human.

In another variation, the one or more bodily tissues or fluids arechosen from blood, urine, saliva, and tears. In a further variation, theone or more bodily tissues or fluids are chosen from liver, muscle,adipose, intestine, brain, and pancreas.

In yet another variation, the water may be partially purified. In afurther variation, the water may be isolated.

In another variation, the method includes the additional step ofmeasuring ²H incorporation into one or more chemical compositions suchas glucose, glycogen, glycerol-triglyceride, triglyceride fatty acid,proteins, and DNA. In a further variation, the chemical composition isglucose. In a still further variation the method includes the additionalstep of measuring endogenous glucose production. In another variation,the method includes the additional step of measuring the proportion oflabeled glucose stored in tissue glycogen relative to sugaradministered. In yet another variation, the method includes theadditional step of measuring the proportion of administered ²H-glucoseundergoing glycolysis.

In another variation, the chemical composition is glycogen.

In another variation, the chemical composition is glycerol-triglyceride.In yet another variation, the method includes the additional step ofcalculating new triglyceride synthesis.

In another variation, the chemical composition is triglyceride fattyacid. In still a further variation, the method includes the additionalstep of calculating new fatty acid synthesis.

In another variation, the method includes the additional step ofcalculating the proportion of labeled fatty acids stored in tissuerelative to labeled fatty acid administered. In a further variation, themethod includes the additional step of calculating the proportion ofadministered labeled fatty acids undergoing fatty acid oxidation.

In another variation, the chemical composition is a protein.

In yet another variation, the chemical composition is DNA. In a furthervariation, the method includes the additional step of calculating therate of DNA synthesis.

In another variation, the method of determining sugar or fatty acidmetabolism in an individual further includes calculating the rate ofincorporation of ²H into the water. In another variation, the methodincludes calculating the rate of incorporation of ²H into one or morechemical compositions such as glucose, glycogen, glycerol-triglyceride,triglyceride fatty acid, proteins, and DNA. Optionally, both the ratesof water formation and chemical composition formation may be monitored.

In another variation, the water may be detected by gaschromatography/mass spectrometry, liquid chromatography-massspectrometry, gas chromatography-pyrolysis-isotope ratio/massspectrometry, or gas chromatography-combustion-isotope ratio/massspectrometry, cycloidal mass spectrometry, Fourier-transform-isotoperatio (IR)-spectroscopy, near IR laser spectroscopy, or isotope ratiomass spectrometry.

In a still further variation, the detecting step may be accomplished bydetecting one part ²H in 10⁷ parts water.

In another aspect, the invention also includes further applications ofthe methods of the invention to determine the metabolism of sugars andfatty acids. In one variation, a drug agent is introduced to theindividual prior to determining the metabolism of one or more sugars orfatty acids, and the effect on an individual is subsequently identified.In another variation, the metabolism determinations are used as asurrogate marker for FDA or other regulatory agency approval of drugs.In yet another variation, the metabolism determination is used for theclinical management of patients. In still a further variation, themetabolism determination includes diagnosing, prognosing, or identifyingindividuals at risk for insulin resistance/diabetes mellitus in theindividual. In another variation, the metabolism determination includesdiagnosing, or identifying individuals at risk for, high-fatdiet-induced obesity. In still another variation, the metabolismdetermination includes monitoring the effects of interventions toprevent or reverse insulin resistance/diabetes mellitus or high-fatdiet-induced obesity. In another variation, the metabolism determinationincludes diagnosing or treating wasting disorders, hypoglycemia, orglycogen storage disease.

The invention is also directed to drug agents that are identified ashaving an effect on the sugar or fatty acid metabolism of theindividual, and isotopically perturbed molecules such as glucose,glycogen, glycerol-triglyceride, triglyceride fatty acid, proteins, andDNA.

The invention is further directed to a kit for determining themetabolism of a sugar in an individual. The kit may include one or morelabeled sugars and instructions for use of the kit. The kit is usefulfor determining sugar metabolism in an individual. The kit may furtherinclude chemical compounds for isolating water. The kit may also includechemical compounds for isolating glucose, glycogen, proteins or DNA. Thekit may also include a tool for administering labeled glucose. The kitmay further include an instrument for collecting a sample from theindividual.

The invention is further directed to a drug agent the effect of whichwas at least partially identified by the methods of the invention.

The invention is further directed to an isotopically perturbed moleculechosen from glycogen, glycerol-triglyceride, triglyceride fatty acid,proteins, and DNA.

The invention is further directed to a method of manufacturing one ormore drug agents at least partially identified by the methods of theinvention.

The invention is further directed to an information storage deviceincluding data obtained from the methods of the invention. The devicemay be a printed report or a computer. The printed report may be printedon paper, plastic, or microfiche. The device may be a computer disc. Thecomputer disk may be chosen from a compact disc, a digital video disc,and a magnetic disc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the fate of ²H attached to fatty acids in the cells. Inthis case, palmitate is shown. The fatty acid is metabolized viaβ-oxidation to release hydrogen atoms from C—H bonds of fats to bodyH₂O. Alternatively, fatty acids may be esterified to producetriglyceride-fatty acids, in this case triglyceride-palmitate.

FIG. 2 depicts the fate of ²H attached to sugars, in this case glucose.Sugars are metabolized via glycolysis and the citric acid cycle torelease hydrogen atoms from C—H bonds of sugars to body H₂O.Alternatively, glucose may form glycogen.

FIG. 3 depicts a schematic molecule of glucose or fat tolerance tests.Glucose or fatty acid metabolism may be measured directly from releaseof ²H to body water. Measurements may include the additional step ofincorporating ²H from body water back into other labeled chemicalcompounds, including labeled glycerol-triglycerides, fattyacid-triglycerides, proteins, DNA, or components thereof.

FIG. 4 depicts a kinetic oral glucose tolerance test in a normal humansubject. The percent glycolysis, measured by deuterium incorporationinto water following administration of deuterium-labeled glucose, isshown over a period of time.

FIG. 5 depicts a kinetic oral glucose tolerance test in a normal mouse.The percent glycolysis, measured by deuterium incorporation into waterfollowing administration of deuterium-labeled glucose, is shown over aperiod of time.

FIG. 6 depicts a kinetic oral glucose tolerance test in a normal rat.The percent glycolysis, measured by deuterium incorporation into waterfollowing administration of deuterium-labeled glucose, is shown over aperiod of time.

DETAILED DESCRIPTION OF THE INVENTION

A method for determining the metabolism of ²H-labeled sugars and fattyacids is described herein. The methods have numerous applications in thefields of medical diagnostics and biological research.

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry,immunology, protein kinetics, and mass spectroscopy, which are withinthe skill of the art. Such techniques are explained fully in theliterature, such as, Molecular Cloning: A Laboratory Manual, secondedition (Sambrook et al., 1989) Cold Spring Harbor Press;Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in MolecularBiology, Humana Press; Cell Biology: A Laboratory Notebook (J. E.Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney,ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: LaboratoryProcedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8)J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.);Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell,eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P.Calos, eds., 1987); Current Protocols in Molecular Biology (F. M.Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mulliset al., eds., 1994); Current Protocols in Immunology (J. E. Coligan etal., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons,1999); and Mass isotopomer distribution analysis at eight years:theoretical, analytic and experimental considerations by Hellerstein andNeese (Am J Physiol 276 (Endocrinol Metab. 39) E1146-E1162, 1999).Furthermore, procedures employing commercially available assay kits andreagents will typically be used according to manufacturer-definedprotocols unless otherwise noted.

II. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, Massisotopomer distribution analysis at eight years: theoretical, analyticand experimental considerations by Hellerstein and Neese (Am J Physiol276 (Endocrinol Metab. 39) E1146-E1162, 1999), herein incorporated byreference. As appropriate, procedures involving the use of commerciallyavailable kits and reagents are generally carried out in accordance withmanufacturer defined protocols and/or parameters unless otherwise noted.

“Metabolism” is used interchangeably with “metabolic fate” and“metabolic consequences,” and refers generally to biosynthesis,breakdown, conversion, oxidation, and/or reduction of sugars and fattyacids.

“Isotopes” refer to atoms with the same number of protons and hence ofthe same element but with different numbers of neutrons (e.g., Hydrogen(H) vs. Deuterium (D)). D is also represented as ²H, as is common in theart.

“Isotopomers” refer to isotopic isomers or species that have identicalelemental compositions but are constitutionally and/or stereochemicallyisomeric because of isotopic substitution, as for CH₃NH₂, CH₃NHD andCH₂DNH₂.

“Isotopologues” refer to isotopic homologues or molecular species thathave identical elemental and chemical compositions but differ inisotopic content (e.g., CH₃NH₂ vs. CH₃NHD in the example above).Isotopologues are defined by their isotopic composition, therefore eachisotopologue has a unique exact mass but may not have a uniquestructure. An isotopologue is usually included of a family of isotopicisomers (isotopomers) which differ by the location of the isotopes onthe molecule (e.g., CH₃NHD and CH₂DNH₂ are the same isotopologue but aredifferent isotopomers).

“Mass isotopomer” refers to a family of isotopic isomers that aregrouped on the basis of nominal mass rather than isotopic composition. Amass isotopomer may comprise molecules of different isotopiccompositions, unlike an isotopologue (e.g., CH₃NHD, ¹³CH₃NH₂, CH₃ ¹⁵NH₂are part of the same mass isotopomer but are different isotopologues).In operational terms, a mass isotopomer is a family of isotopologuesthat are not resolved by a mass spectrometer. For quadrupole massspectrometers, this typically means that mass isotopomers are familiesof isotopologues that share a nominal mass. Thus, the isotopologuesCH₃NH₂ and CH₃NHD differ in nominal mass and are distinguished as beingdifferent mass isotopomers, but the isotopologues CH₃NHD, CH₂DNH₂,¹³CH₃NH₂, and CH₃ ¹⁵NH₂ are all of the same nominal mass and hence arethe same mass isotopomers. Each mass isotopomer is therefore typicallycomposed of more than one isotopologue and has more than one exact mass.The distinction between isotopologues and mass isotopomers is useful inpractice because all individual isotopologues are not resolved usingquadrupole mass spectrometers and may not be resolved even using massspectrometers that produce higher mass resolution, so that calculationsfrom mass spectrometric data must be performed on the abundances of massisotopomers rather than isotopologues. The mass isotopomer lowest inmass is represented as M₀; for most organic molecules, this is thespecies containing all ¹²C, ¹H, ¹⁶O, ¹⁴N, etc. Other mass isotopomersare distinguished by their mass differences from M₀ (M₁, M₂, etc.). Fora given mass isotopomer, the location or position of isotopes within themolecule is not specified and may vary (i.e., “positional isotopomers”are not distinguished).

“Mass isotopomer pattern” refers to a histogram of the abundances of themass isotopomers of a molecule. In one embodiment, the pattern ispresented as percent relative abundances where all of the abundances arenormalized to that of the most abundant mass isotopomer; the mostabundant isotopomer is said to be 100%. In another embodiment, the formfor applications involving probability analysis is proportion orfractional abundance, where the fraction that each species contributesto the total abundance is used (see below). The term isotope pattern issometimes used in place of mass isotopomer pattern, although technicallythe former term applies only to the abundance pattern of isotopes in anelement.

An “individual” refers to a vertebrate animal including a mammal andfurther including a human.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual. The definition encompasses blood and other liquidsamples of biological origin, that are accessible from an individualthrough sampling by minimally invasive or non-invasive approaches (e.g.,urine collection, blood drawing, needle aspiration, and other proceduresinvolving minimal risk, discomfort or effort). The definition alsoincludes samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components, such as proteins or polynucleotides.The term “biological sample” also encompasses a clinical sample such asserum, plasma, other biological fluid, or tissue samples, and alsoincludes cells in culture, cell supernatants and cell lysates.

“Biological fluid” includes but is not limited to urine, blood, bloodserum, amniotic fluid, interstitial fluid, edema fluid, saliva, lacrimalfluid, inflammatory exudates, synovial fluid, abscess, empyema or otherinfected fluid, cerebrospinal fluid, sweat, pulmonary secretions(sputum), seminal fluid, feces, bile, intestinal secretions,conjunctival fluid, tears, vaginal fluid, stool, or other bodily fluid.

“Sugar” refers to a monosaccharide or a polysaccharide comprised ofmonosaccharide residues. Examples of monosaccharides include, but arenot limited to, glucose (both D-glucose and L-glucose), mannose,fructose galactose and sugar derivatives such as glucoronic acid,glucosamine. Examples of polysaccharides include, but are not limitedto, disaccharides such as sucrose, maltose and lactose and longer chainsugar molecules such as glycogen.

“Labeled sugar” refers to a sugar incorporating one or more ²H isotopes.

“Labeled fatty acid” refers to a fatty acid incorporating one or more ²Hisotopes. “Deuterated water” refers to water incorporating one or more²H isotopes. “Labeled glucose” refers to glucose labeled with one ormore ²H isotopes. Specific examples of labeled glucose or ²H-labeledglucose include [6,6-²H₂]glucose, [1-²H₁]glucose, and[1,2,3,4,5,6-²H₇]glucose.

“Partially purifying” refers to methods of removing one or morecomponents of a mixture of other compounds. For example, “partiallypurifying one or more proteins or peptides” refers to removing one ormore proteins or peptides from a mixture of one or more proteins orpeptides or other compounds. As another example, “partially purifyingwater” refers to removing one or more molecules, such as macromolecules,types of macromolecules, or salts, from water.

“Isolating” refers to separating one compound from a mixture ofcompounds. For example, “isolating one or more proteins or peptides”refers to separating one protein or peptide from a mixture of one ormore proteins or peptides or other compounds. “Isolating water” refersto removing all additional compounds beyond trace levels from water.

“Drug agent,” “pharmaceutical agent,” “pharmacological agent,” and“pharmaceutical” are used interchangeably to refer to any chemicalentities, known drug or therapy, approved drug or therapy, biologicalagent (e.g., gene sequences, poly or monoclonal antibodies, cytokines,and hormones). Drug agents include, but are not limited to, any chemicalcompound or composition disclosed in, for example, the 13th Edition ofThe Merck Index (a U.S. publication, Whitehouse Station, N.J., USA),incorporated herein by reference in its entirety.

“Isotopically perturbed” refers to the state of an element or moleculethat results from the explicit incorporation of an element or moleculewith a distribution of isotopes that differs from the distribution thatis most commonly found in nature, whether a naturally less abundantisotope is present in excess (enriched) or in deficit (depleted).

“At least partially identified” in the context of drug discovery anddevelopment means at least one clinically relevant pharmacologicalcharacteristic of a drug agent has been identified using one or more ofthe methods of the present invention. This characteristic may be adesirable one, for example, increasing or decreasing molecular fluxrates through a metabolic pathway that contributes to a disease process,altering signal transduction pathways or cell surface receptors thatalter the activity of metabolic pathways relevant to a disease,inhibiting activation of an enzyme and the like. Alternatively, apharmacological characteristic of a drug agent may be an undesirable onefor example, the production of one or more toxic effects. There are aplethora of desirable and undesirable characteristics of drug agentswell known to those skilled in the art and each will be viewed in thecontext of the particular drug agent being developed and the targeteddisease. Of course, a drug agent can be more than at least partiallyidentified when, for example, when several characteristics have beenidentified (desirable or undesirable or both) that are sufficient tosupport a particular milestone decision point along the drug developmentpathway. Such milestones include, but are not limited to, pre-clinicaldecisions for in vitro to in vivo transition, pre-IND filing go/no godecision, phase I to phase II transition, phase II to phase IIItransition, NDA filing, and FDA approval for marketing. Therefore, “atleast partially” identified includes the identification of one or morepharmacological characteristics useful in evaluating a drug agent in thedrug discovery/drug development process. A pharmacologist or physicianor other researcher may evaluate all or a portion of the identifieddesirable and undesirable characteristics of a drug agent to establishits therapeutic index. This may be accomplished using procedures wellknown in the art.

“Manufacturing drug agents” in the context of the present inventionincludes any means, well known to those skilled in the art, employed forthe making of a drug agent product. Manufacturing processes include, butare not limited to, medicinal chemical synthesis (i.e., syntheticorganic chemistry), combinatorial chemistry, biotechnology methods suchas hybridoma monoclonal antibody production, recombinant DNA technology,and other techniques well known to the skilled artisan. Such a productmay be a final drug agent that is marketed for therapeutic use, acomponent of a combination product that is marketed for therapeutic use,or any intermediate product used in the development of the final drugagent product, whether as part of a combination product or a singleproduct.

“Elevated risk” is used interchangeably herein with “increased risk” andmeans an increase, beyond measurable background levels, of the risk ofan individual for acquiring a condition or disease based on the presenceor absence of one or more risk factors.

“Risk factor” as used herein, means an external or internal factor thatis associated with a disease or disorder. A risk factor may reflect anaspect of causation, whether direct or indirect, but is not so limited.A risk factor may have an association with the onset of a disease ordisorder and may be predictive of such (i.e., a marker of disease), butmay or may not be an indicator of the underlying pathology of thedisease or disorder.

III. Methods of the Invention

Non-invasive tests for determining the metabolism of metabolites such assugars and fatty acids in the body have great utility for clinicaldiagnostics and biomedical research. We disclose here methods that allowhigh-throughput, inexpensive and simple measurements of the disposalpathways and metabolic consequences of sugars and fatty acids in livingorganisms, including humans. As used herein “metabolism,” “metabolicfate” and “metabolic consequences” are used interchangeably and refergenerally to biosynthesis, breakdown, conversion, oxidation, and/orreduction of sugars or fatty acids upon administration. The testinvolves determining the metabolism of sugars and fatty acids byadministering one or more isotope labeled sugars or labeled fatty acidsto an individual, then detecting the release of the label in bodilytissues or fluids to determine the metabolism of the one or more sugarsor fatty acids in the individual. In one embodiment, the test involvesadministration of deuterium-labeled glucose or fatty acids to a humansubject or experimental animal, then measurement of the release ofdeuterium to body water. Highly sensitive measurements of labelenrichments in chemical compositions contained in the bodily tissues orfluids allow great sensitivity and accuracy by this approach.

The tests disclosed herein have utility as drug discovery tools (e.g.,for identifying genes and drugs that alter tissue glucose or fatutilization pathways); as surrogate biomarkers for FDA approval of drugs(e.g., agents influencing fat oxidation or insulin sensitivity oftissues); and as diagnostic measures for the clinical management ofpatients. The methods may be used to diagnose, or identify, the risk ofinsulin resistance or diabetes mellitus. The methods may also be used toidentify diet-induced obesity or the risk of acquiring diet-inducedobesity. The methods may further be used to diagnose or treat wastingdiseases and disorders. Further, the methods may also be used toidentify hypoglycemia or hyperglycemia. In addition, the methods may beused to diagnose or treat glycogen storage diseases. By measuring thetotal disappearance of glucose (D_(glucose)) and the formation ofglycolysis (as described, infra), the rate of glycogen synthesis and/orthe concentration (i.e., the amount) of glycogen synthesized (i.e.,formed) can then be determined. Knowing the rate of glycogen synthesisand/or the amount of glycogen formed, for example, enables the clinicianto evaluate the efficacy of drug agents intended to improve tissueinsulin sensitivity (e.g., in pre-diabetic individuals) or in treatingglycogen storage diseases. Alternatively, knowing the rate of glycogensynthesis and/or the amount of glycogen formed allows the clinician tomore accurately diagnose or prognose a glycogen storage disease.Additionally, the rate of glycogen synthesis and/or the amount ofglycogen formed is a well-accepted early marker for an elevated risk ofdeveloping cardiovascular disease or insulin-resistant disorders such astype II diabetes.

The invention disclosed herein combines the simplicity of an OGTT or fattolerance test with the precision, accuracy and metabolic specificity ofdeuterium tracing. Partitioning labeled ²H, attached to specific C—Hbonds of administered compounds such as sugars or fatty acids, canreveal the specific metabolic fate of the nutrient in a living organismand can be monitored in a high-throughput, inexpensive manner.

Methods of Determining the Metabolism of Compositions Containing Sugarsor Fatty Acids in an Individual

i) Administering Labeled Metabolites to an Individual

a. Compositions Containing Sugars

Compositions containing sugars may include monosaccharides,polysaccharides, or other compounds that are covalently bonded tomonosaccharides or polysaccharides.

²H-labeled sugars may be administered to an individual asmonosaccharides or as polymers of monosaccharide residues. Labeledmonosaccharides may be readily obtained commercially (for example,Cambridge Isotopes, Massachusetts).

Relatively low quantities of compositions that contain ²H-labeled sugarsneed to be administered. Quantities may be on the order of milligrams,10¹ mg, 10² mg, 10³ mg 10⁴ mg, 10⁵ mg, or 10⁶ mg. ²H-labeled sugarenrichment may be maintained for weeks or months in humans and inanimals without any evidence of toxicity. The lower cost of commerciallyavailable labeled monosaccharides, and low quantity that need to beadministered, allow maintenance of enrichments at low expense.

In one particular variation, the labeled sugar is glucose. FIG. 2 showsthe fate of ²H-labeled glucose. Glucose is metabolized by glycolysis andthe citric acid cycle. Glycolysis releases most of the H-atoms from C—Hbonds of glucose; oxidation via the citric acid cycle ensures that allH-atoms are released to H₂O. In a further variation, the labeled glucosemay be [6,6-²H₂]glucose, [1-²H₁]glucose, and [1,2,3,4,5,6-²H₇]glucose.

In another variation, labeled sugar may be fructose or galactose.Fructose is metabolized via the fructose 1-phosphate pathway, andsecondarily through phosphorylation to fructose 6-phosphate byhexokinase. Galactose is metabolized via the galactose to glucoseinterconversion pathway.

Any other sugar may be utilized in the disclosed methods. Othermonosaccharides, include, but are not limited to, trioses, pentoses,hexose, and higher order monosaccharides. Monosaccharides furtherinclude, but are not limited to, aldoses and ketoses.

In another variation, compositions containing polysaccharides may beadministered. The polymers may be formed from monosaccharides: Forexample, labeled glycogen, a polysaccharide, is formed by glucoseresidues. In another variation, labeled polysaccharides may beadministered. As further variation, labeled sugar monomers may beadministered as a component of sucrose (glucose α-(1, 2)-fructose),lactose (galactose β-(1, 4)-glucose), maltose (glucose α-(1,4)-glucose), starch (glucose polymer), or other polymers.

In another variation, the labeled sugar may be administered orally, bygavage, intraperitoneally, intravascularly including intra-arteriallyand intravenously, subcutaneously, or other bodily routes. Inparticular, the sugars may be administered to an individual orally,optionally as part of a food or drink. By “administering” or“administration” is meant any method that introduces the labeled sugarto, in or on an individual.

The individual may be a mammal. In one embodiment, the individual may bean experimental mammal. In another embodiment, the individual may be arodent, primate, hamster, guinea pig, dog, or pig. In yet anotherembodiment, the individual may be a human.

b. Labeled Fatty Acids

Determining the metabolism of compounds that contain ²H-labeled fattyacids is also included in this invention.

²H-labeled fatty acids may be administered to an individual as fats orother compounds containing the labeled fatty acids. ²H-labeled fattyacids may be readily obtained commercially. Relatively low quantities oflabeled fatty acids need to be administered. Quantities may be on theorder of milligrams, 10¹ mg, 10² mg, 10³ mg, 10⁴ mg, 10⁵ mg, or 10⁶ mg.Fatty acid enrichment, particularly with ²H, may be maintained for weeksor months in humans and in animals without any evidence of toxicity. Thelower cost of commercially available labeled fatty acids, and lowquantity that need to be administered, allow maintenance of enrichmentsat low expense.

FIG. 1 shows the fate of ²H-labeled fatty acids during β-oxidation(metabolism) of fatty acids in cells. β-oxidation releases hydrogenatoms from C—H bonds of fats to body H₂O. All H-atoms are released from²H-fatty acids during β-oxidation and, once β-oxidation starts on afatty acid, the process goes to completion. The release of labeled fattyacids, particularly ²H-fatty acid, to labeled water, particularly ²H₂O,accurately reflects fat oxidation. Administration of modest amounts oflabeled-fatty acid is sufficient to measure release of labeled hydrogenor oxygen to water. In particular, administration of modest amounts of²H-fatty acid is sufficient to measure release of ²H to deuterated water(i.e., ²H₂O).

Relatively low quantities of labeled fatty acid or fatty acid residueneed to be administered. Quantities may be on the order of milligrams,10¹ mg, 10² mg, 10³ mg, 10⁴ mg, 10⁵ mg, or 10⁶ mg. ²H-labeled fatty acidenrichment may be maintained for weeks or months in humans and inanimals without any evidence of toxicity. The lower expense ofcommercially available labeled fatty acids and fatty acid residues, andlow quantity that need to be administered, allow maintenance ofenrichments at low expense.

In another variation, the labeled fatty acids may be administeredorally, by gavage, intraperitoneally, intravascularly includingintra-arterially and intravenously, subcutaneously, or other bodilyroutes. In particular, the labeled fatty acids may be administered to anindividual orally, optionally as part of a food or drink. By“administering” or “administration” is meant any method that introducesthe labeled fatty acid to, in or on an individual.

The individual may be a mammal. In one embodiment, the individual may bean experimental mammal. In another embodiment, the individual may be arodent, primate, hamster, guinea pig, dog, or pig. In yet anotherembodiment, the individual may be a human.

(ii) Obtaining One or More Bodily Tissues or Fluids from Said Individual

A biological sample is obtained from bodily tissues or fluids of anindividual. Specific methods of obtaining biological samples are wellknown in the art. Bodily fluids include, but are not limited to, urine,blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid,saliva, tears, vaginal fluid, stool, seminal fluid, and sweat. Thefluids may be isolated by standard medical procedures known in the art.Bodily tissues include, but are not limited to, liver, muscle, adipose,intestine, brain, and pancreas.

In one variation, water may be partially purified. In another variation,the water may be isolated.

In another variation, the one or more bodily tissue or fluids may beobtained after a period of time. In a further variation, the one or morebodily tissues or fluids may be obtained multiple times.

iii) Detecting the Incorporation of ²H into Water

a. Mass Spectrometry

The isotope label, or alternatively, the labeled chemical compositions,may be determined by various methods such as mass spectrometry,particularly gas chromatography-mass spectrometry (GC-MS). Incorporationof labeled isotopes into chemical compositions may be measured directly.Alternatively, incorporation of labeled isotopes may be determined bymeasuring the incorporation of labeled isotopes into one or morehydrolysis or degradation products of the chemical composition. Thehydrolysis or degradation products may optionally be measured followingeither partial purification or isolation by any known separation method,as described previously.

Mass spectrometers convert components of a sample into rapidly movinggaseous ions and separate them on the basis of their mass-to-chargeratios. The distributions of isotopes or isotopologues of ions, or ionfragments, may thus be used to measure the isotopic enrichment in one ormore chemical compositions, or chemical or biochemical degradationproducts.

Generally, mass spectrometers comprise an ionization means and a massanalyzer. A number of different types of mass analyzers are known in theart. These include, but are not limited to, magnetic sector analyzers,electrostatic analyzers, quadrupoles, ion traps, time of flight massanalyzers, and fourier transform analyzers. In addition, two or moremass analyzers may be coupled (MS/MS) first to separate precursor ions,then to separate and measure gas phase fragment ions.

Mass spectrometers may also include a number of different ionizationmethods. These include, but are not limited to, gas phase ionizationsources such as electron impact, chemical ionization, and fieldionization, as well as desorption sources, such as field desorption,fast atom bombardment, matrix assisted laser desorption/ionization, andsurface enhanced laser desorption/ionization.

In addition, mass spectrometers may be coupled to separation means suchas gas chromatography (GC) and high performance liquid chromatography(HPLC). In gas-chromatography mass-spectrometry (GC/MS), capillarycolumns from a gas chromatograph are coupled directly to the massspectrometer, optionally using a jet separator. In such an application,the gas chromatography (GC) column separates sample components from thesample gas mixture and the separated components are ionized andchemically analyzed in the mass spectrometer.

Various mass spectrometers and combinations of separation technologiesand mass spectrometers are contemplated for use in the inventionincluding, but not limited to, gas chromatography/mass spectrometry,liquid chromatography-mass spectrometry, gaschromatography-Pyrolysis-isotope ratio/mass spectrometry, or gaschromatography-combustion-isotope ratio/mass spectrometry, cycloidalmass spectrometry, Fourier-transform-isotope ratio (IR)-spectroscopy,near IR laser spectroscopy, or isotope ratio mass spectrometry

b. Metabolism

Very low quantities of labeled water may be detected. In one embodiment,1 part in 10³ labeled water may be identified. In another embodiment, 1part in 10⁴ labeled water may be identified. In another embodiment, 1part in 10⁵ labeled water may be identified. In another embodiment, 1part in 10⁶ labeled water may be identified. In another embodiment, 1part in 10⁷ labeled water may be identified.

1. Detecting Water Following Sugar Metabolism

The methods of measuring the consequences of sugar ingestion may beaccomplished by measuring sugar metabolism products. The rate ofmetabolic water production from the oxidation of fuels, includingsugars, is sufficient to achieve relatively high levels of labeled waterwhen modest doses of compounds containing labeled sugars areadministered.

Alternatively, labeled glucose may be polymerized to form labeled,glycogen, which may then be measured.

2. Detecting Water Following Fatty Acid Metabolism

The methods of measuring the consequences of fatty acid ingestion may beaccomplished by measuring fatty acid metabolism products. The rate ofmetabolic water production from fatty acid oxidation (metabolism) issufficient to achieve relatively high levels of labeled water,particularly ²H₂O, when modest doses of labeled fatty acids or compoundscontaining fatty acid residues are administered.

FIG. 3 depicts the fatty acid metabolism pathway using deuterium²H-labeled fatty acids. Fatty acids ingested by an individual aredelivered to tissues, optionally stored as triacyl-glycerol, orconverted to water by β-oxidation. Labeled water may then be returned tothe blood stream, and incorporated into bodily fluids.

Labeled water may then be detected to determine the degree of labelincorporation.

iv) The Additional Step of Measuring ²H Incorporated into One or MoreChemical Compositions

The invention also contemplates the additional step of measuring ²Hincorporated into one or more chemical compositions in addition towater. Incorporation of labeled water generated from either labeledglucose or labeled fatty acid metabolism, can be used to measure othersynthesis and storage pathways in an organism (FIGS. 1 and 2). Thesepathways include protein synthesis, lipid synthesis (triglyceridesynthesis and cholesterogenesis), new fat synthesis (de novolipogenesis), and DNA synthesis (cell proliferation). The addition ofthese supplemental measurements (FIG. 3) adds further information to the²H-fatty acid or ²H-glucose labeling strategies.

One or more chemical compositions may be obtained, and optionallypartially purified or isolated, from the biological sample usingstandard biochemical methods known in the art. Chemical compositionsinclude, but are not limited to, glucose, glycogen,glycerol-triglyceride, triglyceride fatty acid, proteins, and DNA.Optionally, fragments of the compositions may also be obtained. Thefrequency of biological sampling can vary depending on differentfactors. Such factors include, but are not limited to, the nature of thechemical composition tested, ease of sampling, and half-life of a drugused in a treatment if monitoring responses to treatment.

In one variation, the one or more chemical compositions may be glucose.In a further variation, the dilution of orally administered labeledsugars, particularly ²H-glucose, in plasma glucose load revealsendogenous glucose production (EGP, FIG. 3). Considerable informationcan be gained about glucose utilization and synthesis pathways in thebody by use of this approach. FIG. 3 depicts the glucose metabolismpathway, specifically for deuterium labeled glucose. Glucose ingested byan individual is delivered to tissues, optionally stored as glycogen, orconverted to water and carbon dioxide via glycolysis and the citric acidcycle. Labeled water, particularly ²H₂O, may then be returned to theblood stream, and incorporated into bodily fluids, then intobiosynthetic products. In a still further variation, the proportion ofglucose may be used to identify the proportion of administered²H-labeled glucose undergoing glycolysis.

In another variation, the method may be used to determine newlysynthesized glycogen. Newly synthesized glycogen can be determinedindirectly by subtracting glycolysis from the total amount of glucoseinitially administered since the total disappearance of glucose is equalto the total amount of glycolysis+the total amount of newly synthesizedglycogen (FIGS. 2 and 3). The following equation can be used tocalculate newly synthesized glycogen:Total glucose−glycolysis=newly synthesized glycogenDetermining newly synthesized glycogen is useful because it is widelybelieved to be an early marker for an elevated risk of insulinresistance, diabetes and cardiovascular disease. That is, the moreglycogen formed from a given amount of glucose administered (as opposedto more glycolysis and less glycogen formed) the higher the risk fordeveloping insulin resistance, diabetes and cardiovascular disease.Glycogen formation is also an early marker for an elevated risk ofcerebrovascular disease.

In another variation, the rate of appearance of glycogen(Ra_(glycogen)), i.e., the rate of glycogen synthesis, may be determinedby subtracting the rate of appearance of glycolysis (Ra_(glycolysis))from the rate of disappearance of glucose (Rd_(glucose)). The followingequation can be used to calculate the rate of new glycogen synthesis:Rd _(glucose) −Ra _(glycolysis) =Ra _(glycogen)

Knowing the rate of appearance of glycogen (i.e., the rate of glycogensynthesis) provides additional useful information as a slower rate ofglycogen synthesis may be associated with an elevated risk of developingdiabetes, cardiovascular disease, and cerebrovascular disease.Furthermore, a slower rate of glycogen synthesis together with anincrease in newly synthesized glycogen may be associated with anelevated risk of developing diabetes, cardiovascular disease, andcerebrovascular disease.

Similarly, knowing glycolysis may also be useful for determining anelevated risk of diabetes, cardiovascular disease, and cerebrovasculardisease. Knowing the rate of glycolysis provides additional informationuseful for determining an elevated risk, as described above for the rateof glycogen synthesis. Glycolysis and the rate of glycolysis may bedetermined by measuring the amount of ²H₂O formed after administrationof ²H-Glucose, as is described supra.

In another variation, the one or more chemical compositions may beglycogen. Glycogen may be measured directly by direct sampling usinginvasive or non-invasive procedures well known in the art. In a furthervariation, the one or more chemical compositions may be triglycerides.In a further variation, the method may be used to determine newtriglyceride synthesis.

In another variation, the one or more chemical compositions may becompounds that include triglyceride-fatty acids. In a further variation,the method may be used to calculate new fatty acid synthesis.

In a still further variation, the method may be used to calculate theratio of labeled fatty acids to stored fatty acids. In a still furthervariation, the method may be used to calculate the proportion ofadministered fatty acids undergoing fatty acid oxidation.

In another variation, the chemical composition may include a protein.

In a further variation, the composition may include DNA. The measurementof DNA incorporation may then be used to determine the rate of new cellproliferation.

The one or more chemical compositions may also be purified, partiallypurified, or optionally, isolated, by conventional purification methodsincluding high performance liquid chromatography (HPLC), fastperformance liquid chromatography (FPLC), gas chromatography, gelelectrophoresis, and/or any other separation methods.

The one or more chemical compositions may be hydrolyzed or otherwisedegraded to form smaller subunits. Hydrolysis or other degradationmethods include any method known in the art, including, but not limitedto, chemical hydrolysis (such as acid hydrolysis) and biochemicalhydrolysis (such as enzyme cleavage or degradation). Hydrolysis ordegradation may be conducted either before or after purifying and/orisolating the one or more chemical compositions. For example, polymersformed of monosaccharides may be degraded to form smaller units ofmultiple monosaccharide residues, and/or optionally, monosaccharideconstituents. Glycogen may be degraded chemically or proteolytically toform polysaccharides formed from glucose residues, or optionally,glucose monomers. Proteins may be chemically or proteolytically degradedto form oligopeptides, or optionally, amino acids. Fatty acids may bedegraded to form ketone bodies, carbon dioxide, and water. DNA may bedegraded to form polynucleotides, oligonucloetides, nucleotides,nucleosides, nucleic acid bases, or nucleic acid backbones. Degradationproducts may be partially purified, or optionally, isolated, byconventional purification methods including high performance liquidchromatography (HPLC), fast performance liquid chromatography (FPLC),gas chromatography, gel electrophoresis, and/or any other methods knownin the art.

v) Calculating Kinetic Parameters

Rates or total amounts of ²H incorporation into water may be calculated.Rates of incorporation into other biopolymers may also be calculated. Inone variation, the rate of incorporation of ²H into water may becalculated. In another variation, the rate of degradation of compoundscontaining labeled sugars or fatty acids may be measured. In a furthervariation, the biosynthesis and degradation rates of biopolymers such asglucose, glycogen, glycerol-triglyceride, triglyceride fatty acid,proteins, and DNA may also be determined. In still another variation,both rates of labeled water formation and biopolymer formation may becalculated. Finally, the rates may be used individually, or incombination, to diagnose, prognose, or identify the risk of metabolic ormetabolically-related diseases or disorders.

Synthesis and degradation rates may be calculated by mass isotopomerdistribution analysis (MIDA), which may be used to calculate thedegradation or biosynthesis rates of metabolites and/or water bymeasuring the production of labeled water. In addition, MIDA may be usedto calculate the synthesis rate of biopolymers such as glucose,glycogen, glycerol-triglyceride, triglyceride fatty acid, proteins, andDNA after the sugar or fatty acid containing metabolites aremetabolized.

Variations of the MIDA combinatorial algorithm are discussed in a numberof different sources known to one skilled in the art. Specifically, theMIDA calculation methods are the subject of U.S. Pat. No. 5,336,686,incorporated herein by reference. The method is further discussed byHellerstein and Neese (1999), incorporated herein by reference, as wellas Patterson and Wolfe (1993), and Kelleher and Masterson (1992).

In addition to the above-cited references, calculation softwareimplementing the method is publicly available from Marc Hellerstein atthe University of California, Berkeley.

In brief, calculation of the number (n) of metabolically exchangedH-atoms between sugars or fatty acids and cellular water was bycombinatorial analysis, or MIDA. The relative fraction of double-labeledto single-labeled sugars or fatty acid molecules reveals n if theprecursor pool enrichment of ²H (p) is known. If one assumes that preflects body labeled water enrichment, then n can be calculated bycombinatorial analysis.

Fractional abundances of mass isotopomers result from mixing naturalabundance molecules with molecules newly synthesized from a pool oflabeled monomers characterized by the parameter p. A mixture of thistype can be fully characterized by f, the fraction new, and p. Thealgorithm proceeds in step-wise fashion, beginning with the simplestcalculation, a molecule synthesized from a single element containingisotopes with the same fractional abundances that occur in nature andnot mixed with any other molecules. We then proceed to moleculescontaining more than one element with all isotopes at natural abundance;then to non-polymeric molecules containing different elements, some ofwhich are in groups whose isotope composition is not restricted tonatural abundance but is variable; then to polymeric moleculescontaining combinations of repeating chemical units (monomers), whereinthe monomers are either unlabeled (containing a natural abundancedistribution of isotopes) or potentially labeled (containing anisotopically-perturbed element group); and finally to mixtures ofpolymeric molecules, composed of both natural abundance polymers andpotentially labeled polymers, the latter containing combinations ofnatural abundance and isotopically-perturbed units.

The last-named calculation addresses the condition generally present ina biological system, wherein polymers newly synthesized during theperiod of an isotope incorporation experiment are present along withpre-existing, natural abundance polymers and the investigator isinterested in determining the proportion of each that is present, inorder to infer synthesis rates or related parameters.

Methods of Use

Using the methods disclosed herein, metabolic consequences of nutrientingestion may be determined for a number of metabolites in anindividual. These consequences may be applied for diagnostic and/ormonitoring uses. There are numerous research and clinical applicationsof this technique.

In one variation, the effect of a drug agent on an individual may bemonitored. A change in the sugar or fatty acid metabolism in anindividual to which a drug agent has been administered identifies thedrug agent as capable of altering the sugar or fatty acid metabolism ofthe individual. The drug agent may be administered to the sameindividual, or different living systems. Drug agents may be any chemicalcompound or composition known in the art. Drug agents include, but arenot limited to, any chemical compound or composition disclosed in, forexample, the 13th Edition of The Merck Index (a U.S. publication,Whitehouse Station, N.J., USA), incorporated herein by reference in itsentirety.

In another variation, drug agents can be at least partially identifiedas to desirable or undesirable (or both) characteristics. Suchinformation is useful in evaluating whether a drug agent should beadvanced in clinical development, for example, whether a drug agentshould be tested in in vivo animal models, whether it should be thesubject of clinical trials, and whether it should be advanced further inthe clinical trial setting (e.g., after an IND filing and/or aftercompletion of phase I, phase II and/or phase III trials). Once advancedthrough the filing and approval of an NDA, it is readily apparent thatthe methods of the present invention allow for the early identificationof drug agents useful in the treatment of metabolic diseases such asdiabetes, cardiovascular disease, and other obesity-related diseases ordisorders. In another embodiment, the fate of nutrients as surrogatesduring FDA trials may be monitored.

In another variation, the methods may be used to identify individuals atrisk for diabetes. In another variation, the methods may be used toidentify patients at risk for high-fat diet-induced obesity.

In another variation, the methods may be used to diagnose, prognose, oridentify the risk of insulin resistance/diabetes mellitus (type IIdiabetes) in an individual. In a further variation, the methods may beused to diagnose, prognose, or identify the risk of high-fatdiet-induced obesity in an individual. In another variation, the methodsmay be used to monitor the effects of interventions or treatment methodsto prevent or reverse insulin resistance/diabetes mellitus or high-fatdiet-induced obesity.

Isotopically-perturbed Molecules

In another variation, the methods provide for the production ofisotopically-perturbed molecules (e.g., labeled fatty acids, lipids,carbohydrates, proteins, nucleic acids and the like). Theseisotopically-perturbed molecules comprise information useful indetermining the flux of molecules within the metabolic pathways ofinterest. Once isolated from a cell and/or a tissue of an organism, oneor more isotopically-perturbed molecules are analyzed to extractinformation as described, supra.

In other variations, the methods may be used to diagnose or treatwasting diseases or disorders, hypoglycemia, or glycogen storagedisease.

Kits

In another aspect, the invention provides kits for analyzing themetabolic fate of glucose or fatty acids in vivo. The kits may includelabeled glucose or fatty acids. The kits may also include chemicalcompounds known in the art for isolating chemical and biochemicalcompounds from urine, bone, or muscle and/or chemicals necessary to geta tissue sample, automated calculation software for combinatorialanalysis, and instructions for use of the kit are optionally included inthe kit.

Other kit components, such as tools for administration of compoundscontaining labeled sugars and fatty acids are optionally included. Toolsmay include measuring cups, needles, syringes, pipettes, IV tubing), mayoptionally be provided in the kit. Similarly, instruments for obtainingsamples from the subject (e.g., specimen cups, needles, syringes, andtissue sampling devices) may also be optionally provided.

Information Storage Devices

The invention also provides for information storage devices such aspaper reports or data storage devices comprising data collected from themethods of the present invention. An information storage deviceincludes, but is not limited to, written reports on paper or similartangible medium, written reports on plastic transparency sheets ormicrofiche, and data stored on optical or magnetic media (e.g., compactdiscs, digital video discs, magnetic discs, and the like), or computersstoring the information whether temporarily or permanently. The data maybe at least partially contained within a computer and may be in the formof an electronic mail message or attached to an electronic mail messageas a separate electronic file. The data within the information storagedevices may be “raw” (i.e., collected but unanalyzed), partiallyanalyzed, or completely analyzed. Data analysis may be by way ofcomputer or some other automated device or may be done manually. Theinformation storage device may be used to download the data onto aseparate data storage system (e.g., computer, hand-held computer, andthe like) for further analysis or for display or both. Alternatively,the data within the information storage device may be printed ontopaper, plastic transparency sheets, or other similar tangible medium forfurther analysis or for display or both. The information storage devicemay provide for retrieval of the data. Such retrieval can be for thepurpose of display and/or for further analysis or for any other purpose.

The following examples are provided to show that the methods of theinvention may be used to determine the fate of metabolic glucose orfatty acids. Those skilled in the art will recognize that while specificembodiments have been illustrated and described, they are not intendedto limit the invention.

EXAMPLES Example 1 Kinetic OGTT—Glycolytic Disposal of Glucose in NormalRats and Mice

The kinetic oral glucose tolerance test for mice and rats is depicted inFIGS. 5 and 6, respectively. The figures depict percent glycolysis,measured by deuterium incorporation into water following administrationof deuterium-labeled glucose.

Sprague-Dawley rats (200-250 g, Simonsen Inc., Gilroy, Calif.) andC57Blk/6ksj mice (10-15 g, Jackson Laboratories, Bar Harbor, Me.) wereused. Housing was in individual cages for rats and groups of 5 for mice.Feeding was ad-libitum with Purina® rodent chow. All studies receivedprior approval from the UC Berkeley Animal Care and Use Committee.

The ²H-glucose labeling protocol consisted of an initial intraperitoneal(ip) injection of 99.9% [6,6-²H₂] glucose. For labeling rats and mice, 2mg labeled glucose per gram body weight were introduced. Body water wascollected as serum at various timepoints.

Glycolysis was measured by measuring deuterium in body water as apercent of administered [6,6-²H₂] glucose normalized to account fordifferent molar quantities of deuterium in molecular glucose andmolecular water. Deuterized water was measured by isotope ratio massspectrometry.

Example 2 Kinetic OGTT—Glycolytic Disposal of Glucose in Normal Rats andMice

A kinetic oral glucose tolerance test in a human subject is depicted inFIG. 4. The figure depicts percent glycolysis, measured by deuteriumincorporation into water following ingestion of deuterium labeledglucose.

The ²H-glucose labeling protocol consisted of an oral ingestion of 99.9%[6,6-²H₂] glucose. 15 grams glucose in 50 grams oral load (30%[6,6-²H₂]) were ingested by the human subject. Body water was collectedas serum at various timepoints.

Glycolysis was measured by measuring deuterium in body water as apercent of administered [6,6-²H₂] glucose normalized to account fordifferent molar quantities of deuterium in molecular glucose andmolecular water. Deuterized water was measured by isotope ratio massspectrometry.

Example 3

[6,6-²H₂] glucose was administered orally (15 grams in water) to a leanmale human subject (Subject #1), to an overweight but not obese malehuman subject (Subject #2), to an obese female human subject (Subject#3), and to a lean male human subject with HIV/AIDS (Subject #4). Bloodsamples were collected (10 cc) every hour for four hours. ²H content ofblood glucose was measured by isolating glucose from blood and preparinginto a form compatible with isotope ratio mass spectrometry. Theisotopic (²H₂O) content of body water was measured by isolating waterfrom the blood and preparing into a form compatible with isotope ratiomass spectrometry. Mass spectrometry was performed to calculate thefraction of ²H from ²H-glucose released into body water. This representsglycolysis/oxidation from the administered glucose load. Measurement of²H-glucose content measured by mass spectrometry was compared toadministered ²H content of administered ²H-glucose to calculate thebody's production rate of glucose. Fasting plasma insulin levels weremeasured by radioimmunoassay (RIA) specific for insulin. RIA kits arereadily available from a variety of commercial sources such as LincoResearch Inc., St. Charles, Mo., USA or Phoenix Pharmaceutical, Inc.,Belmont, Calif., USA. Plasma glucose levels were measured by the use ofglucose oxidase, a technique well known in the art. Kits containingglucose oxidase for the measurement of glucose are readily available,for example from Sigma Aldrich, St. Louis, Mo., USA. Table 1 depicts theresults.

TABLE 1 Subjects Undergoing ²H—OGTT (75 g glucose; 15 g [6,6-²H]Glucose) Fasting Glycolysis Plasma Peak Plasma (mMoles²H₂O Subject BMIInsulin Glucose produced) # (kg/m²) Gender (μU/mL) (mg/dL) 2 h 4 h 123.5 M <15 105 25 50 2 27.2 M 20 96 26 41 3 31.8 F 33 108 15 34 4 23.5M >30 225 16 33 BMI = Body Mass Index Plasma glucose > 200 mg/dL during²H—OGTT indicates glucose intolerance or diabetes Plasma insulin > 20indicates hyperinsulinemia/insulin resistance Maximal production of ²H₂Ofrom 15 g ²H-glucose is 82 × 10⁻³ moles

As can be seen from table 1, supra, Subject #1 is a normal, lean healthymale subject (normal control). Subject #2 is a normal, overweight butnot obese healthy male subject. Subject #3 is a normal, obese healthyfemale. Subject #4 is a lean male with HIV/AIDS. The data identifiesclinically evident glucose intolerant or diabetic individuals despitethe absence of obesity (Subject #4). Subject #4 is an HIV positive malewith AIDS receiving protease-inhibitor containing anti-retroviraltherapy. People with AIDS who receive anti-retroviral treatments oftendevelop a glucose intolerant or diabetic phenotype. The data in table 1also identifies pre-diabetic (insulin resistant) individuals beforeglucose intolerance is apparent (Subject #3). This method allows forearly detection of glucose intolerance.

REFERENCES

-   1. Veerkamp J H, Van Moerkerk H T B, Glatz J F C, Zuurveld J G E M,    Jacobs A E M, Wagenmakers A J M. ¹⁴CO₂ production is no adequate    measure of ¹⁴C-fatty acid oxidation. Biochem Med Metab Biol    35:248-59, 1986.-   2. Malewiak M I, Griglio S, Kalopissis A D, LeLiepure X. Oleate    metabolism in isolated hepatocytes from lean and obese Zucker rats.    Influence of a high-fat diet and in vitro response to glucagon.    Metab Clin Exp 32:661-8, 1993.-   3. Van Hinsbergh V, Veerkamp J H, van Moerkerk H T B. Palmitate    oxidation by rat skeletal muscle mitochondria. Comparison of    polarographic and radiochemical experiments Arch Biochem Biophys    190:762, 1978.-   4. Hellerstein M K. Methods for measurement of fatty acid and    cholesterol metabolism. In: Howard B, Packard C, eds. Current    Opinion in Lipidology 6:172-81, 1995.-   5. Katz, J., and R. Rognstad. Futile cycles in the metabolism of    glucose. In: Current Topics in Cellular Regulation. Vol 10, edited    by B. Horecker and E. Stadman. New York: Academic Press, 1976, p.    238-239.-   6. Rossetti L, Lee Y T, Ruiz J, Aldridge S C, Shamoon H, Boden G.    Quantitation of glycolysis and skeletal muscle glycogen synthesis in    humans. Am J Physiol 265:E761-9, 1993.-   7. Turner S, Neese R A, Murphy E, Antelo F, Thomas T, Hellerstein    M K. Measurement of triglyceride synthesis and turnover in vivo by    ²H₂O incorporation into the glycerol moiety and application of mass    isotopomer distribution analysis. Am J Physiol, Submitted, 2002.-   8. Neese R A, Misell L, Antelo F, Hoh R, Chu A, Strawford A,    Christiansen M, Hellerstein M K. Measurement of DNA synthesis in    slow turnover cells in vivo using ²H₂O labeling of the deoxyribose    moiety: application to human adipocytes. Proc Natl Acad Sci USA, In    Press, 2002.-   9. Misell L, Thompson J, Antelo F, Chou Y-C, Nandi S, Neese R,    Hellerstein M K. A new in vivo stable isotope method using ²H₂O for    measuring mammary epithelial cell proliferation. FASEB J 14(4):A786,    2000.-   10. Kim J, Neese R, Hellerstein M K. A new method to measure    proliferation rates of colon epithelial cells. FASEB J 14(4):A718,    2000.-   11. Antelo F, Strawford A, Neese R A, Christiansen M, Hellerstein M.    Adipose triglyceride (TG) turnover and de novo lipogenesis (DNC) in    humans: measurement by long-term ²H₂O labeling and mass isotopomer    distribution analysis (MIDA). FASEB J 16:A400, 2002.-   12. Chu A, Cesar D, Ordonez E, Hellerstein M. An in vivo method for    measuring vascular smooth muscle cell (VSMC) proliferation using    ²H₂O. Circulation, 2000.-   13. Hellerstein M K, Neese R A, Kim Y-K, Schade-Serin V, Collins M.    Measurement of synthesis rates of slow-turnover proteins from ²H₂O    incorporation into non-essential amino acids (NEAA) and application    of mass isotopomer distribution analysis (MIDA). FASEB J 16:A256,    2002.-   14. Kim Y-K, Neese R A, Schade-Serin V, Collins M, Misell L,    Hellerstein M K. Measurement of synthesis rates of slow-turnover    proteins based on ²H₂O incorporation into non-essential amino acids    and application of mass isotopomer distribution analysis.    Biochemical J, Submitted, 2002.-   15. Reaven G M. Banting lecture 1988. Role of insulin resistance in    human disease. Diabetes 37(12):1595-607, 1988.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes tothe same extent as if each individual publication, patent, or patentapplication were specifically and individually indicated to be soincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it is readily apparent to those of ordinaryskill in the art in light of the teachings of this invention thatcertain changes and modifications may be made thereto without departingfrom the spirit and scope of the claims.

Applicants have not abandoned or dedicated to the public any unclaimedsubject matter.

1. A method of determining a rate of in vivo metabolism of one or moresugars or fatty acids in an individual, said method comprising: (a)administering a known quantity of ²H-labeled sugars or ²H-labeled fattyacids to an individual for a sufficient time for said ²H-labeled sugarsor ²H-labeled fatty acids to be metabolized in said individual toproduce ²H-labeled water; (b) obtaining one or more bodily tissues orfluids at one or more times from said individual, wherein said one ormore bodily tissues or fluids comprise a portion of said ²H-labeledwater; (c) determining a quantity of ²H present in said portion of said²H-labeled water to quantify an amount of ²H released into body water;and (d) calculating a ratio of said amount of ²H released into bodywater to said known amount of ²H administered to determine said rate ofin vivo metabolism of said one or more of sugars or fatty acids in saidindividual.
 2. The method according to claim 1, wherein said knownquantity of ²H-labeled sugars comprise ²H-labeled glucose.
 3. The methodaccording to claim 2, wherein said ²H-labeled glucose is selected fromthe group consisting of [6,6-²H₂]glucose, [1-²H₁]glucose, and[1,2,3,4,5,6-²H₇]glucose.
 4. The method according to claim 1, whereinsaid known quantity of ²H-labeled sugars or ²H-labeled fatty acids isadministered by a technique selected from the group consisting of oral,gavage, intraperitoneal, intravascular, and subcutaneous administration.5. The method according to claim 4, wherein said known quantity of²H-labeled sugars or ²H-labeled fatty acids is administered orally. 6.The method according to claim 1, wherein said individual is a mammal. 7.The method according to claim 6, wherein said mammal is selected fromthe group consisting of human, rodent, primate, dog, and pig.
 8. Themethod according to claim 7, wherein said mammal is a human.
 9. Themethod according to claim 1, wherein said one or more bodily tissues orfluids are selected from the group consisting of blood, urine, saliva,and tears.
 10. The method of claim 1, wherein said one or more bodilytissues or fluids are selected from the group consisting of liver,muscle, adipose, intestine, brain, and pancreas.
 11. The method of claim1, comprising the additional step of partially purifying said portion ofsaid ²H-labeled water from said one or more bodily tissues or fluids.12. The method of claim 11, comprising the additional step of isolatingsaid portion of said ²H-labeled water from said one or more bodilytissues or fluids.
 13. The method according to claim 1, comprising theadditional step of calculating a proportion or storage rate ofadministered ²H-labeled fatty acids by calculating the proportion of²H-labeled fatty acids not metabolized in said individual to produce²H-labeled water.
 14. The method according to claim 1, wherein saidquantity of ²H present in said portion of said ²H-labeled water isdetermined by a method selected from the group consisting of gaschromatography/mass spectrometry, liquid chromatography-massspectrometry, gas chromatography-pyrolysis-isotope ratio/massspectrometry, gas chromatography-combustion-isotope ratio/massspectrometry, cycloidal mass spectrometry, Fourier-transform-isotoperatio (IR)-spectroscopy, near IR laser spectroscopy, and isotope ratiomass spectrometry.
 15. The method according to claim 1, wherein saiddetermining step is accomplished by determining one part ²H in 10⁷ partswater.
 16. The method according to claim 1, wherein said rate of in vivometabolism is used as a surrogate marker for FDA approval of drugs. 17.The method according to claim 1, wherein said rate of in vivo metabolismis used for clinical management of patients.
 18. The method according toclaim 1, wherein said method further comprises identifying individualsat risk for insulin resistance and diabetes mellitus.
 19. The methodaccording to claim 1, wherein said method further comprises diagnosinghigh-fat diet-induced obesity.
 20. The method according to claim 1,wherein said method further comprises identifying individuals at riskfor high-fat diet-induced obesity.
 21. The method according to claim 1,wherein said method further comprises a step of monitoring the effectsof interventions to prevent or reverse insulin resistance, diabetesmellitus, and high-fat diet-induced obesity.
 22. The method according toclaim 1, further comprising a step of diagnosing or treating wastingdisorders.
 23. The method according to claim 1, further comprising astep of diagnosing or treating hypoglycemia.
 24. The method according toclaim 1, further comprising a step of diagnosing or treating glycogenstorage disease.
 25. The method according to claim 1, comprising theadditional step of calculating a proportion or storage rate ofadministered ²H-labeled sugars by calculating the proportion of²H-labeled sugars not metabolized in said individual to produce²-labeled water.